The small GTPase EhRabB of Entamoeba histolytica is differentially expressed during phagocytosis

Abstract

It has been described that the pathogenicity of Entamoeba histolytica is influenced by environmental conditions and that transcription profile changes occur during invasion, suggesting that gene expression may be involved in the virulence of this parasite. However, the molecular mechanisms that are implicated in the control of gene expression in this microorganism are poorly understood. Here, we showed that the expression of the EhRabB protein, a small GTPase involved in phagocytosis, is modified through the interaction with red blood cells. By ELISA, Western blot, and immunofluorescence assays, we observed that the expression of EhRabB diminished after 5 min of the interaction of trophozoites with red blood cells, but protein level was recovered at subsequent times. In the EhRabB amino acid sequence, we found two lysine residues that could be target for ubiquitin modification and trigger the degradation of this GTPase at early times of phagocytosis. The analysis of the expression of the EhrabB mRNA showed that the interaction of trophozoites with red blood cells produces a drastic diminishing in its half-life. In addition, promoter assays using the chloramphenicol acetyltransferase reporter gene and electrophoretic mobility shift assays experiments showed that the URE1 motif located in the promoter region of EhrabB is involved in the expression regulation of this gene during phagocytosis. Moreover, the immunolocalization of the URE1-binding protein during phagocytosis indicated that the transcription of the EhrabB gene is determined, at least in part, by the translocation of this transcription factor to nuclei. These results suggested that the expression of particular genes of this parasite is controlled at several stages.

EhRabB mobilises the EhCPADH complex through the actin cytoskeleton during phagocytosis of Entamoeba histolytica

“…However, these experiments proven slight changes in the amount of EhRabB protein (Figure ). In contrast to these results, other authors found that EhRabB protein diminished after 5 min of phagocytosis, and later, it augmented to almost reach the basal level (Hernandes‐Alejandro et al, ). Here, to obtain a better resolution in immunofluorescence assays, we drastically diminished the trophozoites: RBCs proportion.…”

“…By this fact, we hypothesised that at least part of the complex is driven for degradation. In contrast, a part of EhRabB could be degraded by other pathways, as it was suggested by Hernandes‐Alejandro et al (). Interestingly, MVB‐like structures presented internal vesicles that could correspond to the intraluminal vesicles.…”

“…Here, to obtain a better resolution in immunofluorescence assays, we drastically diminished the trophozoites: RBCs proportion. Whereas Hernandes‐Alejandro et al () used 1:100 trophozoites per RBCs, we used 1:25. In fact, in our laboratory, we have experimentally probed that the trophozoites, RBCs ratio can alter the rate of phagocytosis, and as we are showing here, the expression of certain proteins could be also altered.…”

Genome-wide identification of pathogenicity factors of the free-living amoeba Naegleria fowleri

“…The Rab GTPase EhRabB, which is localized in cytoplasmic vesicles, is involved in the phagocytosis of E. histolytica [37, 38]. Rho family GTPases, including Rab proteins such as EhRabB, regulate the cytoskeleton and associated pathogenic processes such as phagocytosis, which in turn, is controlled by vesicular trafficking [39].…”

EhRab21 mobilization during erythrophagocytosis in Entamoeba histolytica

“…Therefore, the subcellular localization of Eh Rab21 during erythrophagocytosis was analyzed. Eh RabB is relocalized during the early steps of erythrophagocytosis from the cytoplasm to the plasma membrane (Hernandes‐Alejandro et al, ). In the case of Eh Rab21, it is not present in erythrocyte‐containing vesicles.…”